Abstract
Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein.
Highlights
Acetylcholine (ACh) and its cholinergic components, e.g. cholinesterases (ChEs), choline acetyltransferase (ChAT), receptors (AChRs) and vesicular transporters are expressed at synapses, but in many non-neural cell types, suggesting synapse-independent roles of cholinergic systems, e.g. regulating development or immune responses [1,2,3,4,5,6]
Already in a HH16 embryo, AChE was detectable with the earliest indication of a limb anlage along a slightly bulged ectodermal surface (Fig 1A, arrow)
By HH19, the wing anlage (Fig 1C and 1D) had further extended, with an outer contour of strong AChE expression on its ventral side (Fig 1C), As shown on AChE-stained cryosection of HH19 (Fig 1D), an ectodermal layer expressed AChE, whereby a graded increase of activity to words the distal end of the limb was evident. This area corresponds with the apical ectodermal ridge (AER), the first organizing center of the vertebrate limb [43]
Summary
Acetylcholine (ACh) and its cholinergic components, e.g. cholinesterases (ChEs), choline acetyltransferase (ChAT), receptors (AChRs) and vesicular transporters (vAChT) are expressed at synapses, but in many non-neural cell types, suggesting synapse-independent roles of cholinergic systems ( known as non-neuronal cholinergic systems, NNCS), e.g. regulating development or immune responses [1,2,3,4,5,6]. Roles of ChEs, e.g. acetyl- (AChE) and butyrylcholinesterase (BChE) in developmental processes should not be underestimated, since they represent—due to their high turnover rate—rate-limiting components within classica
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